Differential PKC-dependent and -independent PKD activation by G protein alpha subunits of the Gq family: Selective stimulation of PKD Ser(748) autophosphorylation by G alpha q

作者：Waldron Richard T^*; Innamorati Giulio; Eugenia Torres Marquez M; Sinnett Smith James; Rozengurt Enrique

来源：Cellular Signalling, 2012, 24(4): 914-921.

DOI：10.1016/j.cellsig.2011.12.014

• G alpha 11
• G alpha 15
• G alpha 14
• AlF4

Protein kinase D (PKD) is activated within cells by stimulation of multiple G protein coupled receptors (GPCR). Earlier studies demonstrated a role for PKC to mediate rapid activation loop phosphorylation-dependent PKD activation. Subsequently, a novel PKC-independent pathway in response to Gag-coupled GPCR stimulation was identified. Here, we examined further the specificity and PKC-dependence of PKD activation using COS-7 cells cotransfected with different Gq-family G alpha and stimulated with aluminum fluoride (AlF4(-)). PKD activation was measured by kinase assays, and Western blot analysis of activation loop sites Ser(744), a prominent and rapid PKC transphosphorylation site, and Ser(748), a site autophosphorylated in the absence of PKC signaling. Treatment with AlF4(-) potently induced PKD activation and Ser744 and Ser748 phosphorylation, in the presence of cotransfected G alpha q, G alpha 11, G alpha 14 or G alpha 15. These treatments achieved PKD activation loop phosphorylation similar to the maximal levels obtained by stimulation with the phorbol ester, PDBu. Preincubation with the PKC inhibitor GF1 potently blocked G alpha 11-, G alpha 14-, and G alpha 15-mediated enhancement of Ser(748) phosphorylation induced by AlF4(-), and largely abolished Ser744 phosphorylation. In contrast, Ser748 phosphorylation was almost completely intact, and Ser744 phosphorylation was significantly activated in cells cotransfected with Gaq. Importantly, the differential Ser748 phosphorylation was also promoted by treatment of Swiss 3T3 cells with Pasteurella multocida toxin, a selective activator of Gag but not Gall. Taken together, our results suggest that Gaq, but not the closely related G alpha 11, promotes PKD activation in response to GPCR ligands in a unique manner leading to PKD autophosphorylation at Ser(748).

• 出版日期2012-4